Light harvesting multichromophore compositions and methods of using the same

ABSTRACT

Light harvesting luminescent multichromophores that are configured upon excitation to transfer energy to, and amplify the emission from, an acceptor signaling chromophore in energy-receiving proximity therewith are provided. Also provided are compositions for labelling a target. The labelling composition may include a donor light harvesting multichromophore and an acceptor signaling chromophore in energy-receiving proximity to the donor light harvesting multichromophore. Also provided is an aqueous composition for labelling a target, including: a donor light harvesting multichromophore; an acceptor signaling chromophore in energy-receiving proximity therewith; and a sensor biomolecule. Methods for using the subject compositions are also provided.

CROSS-REFERENCING

This patent application is a continuation-in-part of: U.S. applicationSer. No. 13/356,500, filed on Jan. 23, 2012 which is a continuation ofU.S. application Ser. No. 11/746,055, filed May 8, 2007, which is acontinuation of U.S. application Ser. No. 10/600,286, filed Jun. 20,2003, which claims the benefit of U.S. Provisional Application No.60/406,266, filed Aug. 26, 2002 and U.S. Provisional Application No.60/390,524, filed on Jun. 20, 2002, which applications are incorporatedherein by reference for all purposes; and this patent application isrelated to and claims the benefit of U.S. application Ser. No.14/460,245, filed on Aug. 14, 2014, which is a continuation of U.S.application Ser. No. 14/086,532, filed Nov. 21, 2013, which is acontinuation of U.S. application Ser. No. 13/544,303, filed Jul. 9,2012, which is a continuation of U.S. application Ser. No. 12/632,734,filed Dec. 7, 2009, which is a continuation of U.S. application Ser. No.11/854,365, filed Sep. 12, 2007, which is a divisional of U.S.application Ser. No. 10/648,945, filed Aug. 26, 2003, which claims thebenefit of U.S. Provisional Application No. 60/406,266, filed Aug. 26,2002; U.S. application Ser. No. 10/779,412, filed on Feb. 13, 2004,which claims the benefit of U.S. Provisional Application No. 60/447,860,filed Feb. 13, 2003; and U.S. application Ser. No. 13/075,172, filed onMar. 29, 2011 which is a continuation of U.S. application Ser. No.11/561,893, filed Nov. 21, 2006, which is a divisional of U.S.application Ser. No. 10/666,333, filed Sep. 17, 2003; which applicationsare incorporated herein by reference for all purposes.

GOVERNMENT SUPPORT

The following invention was made with support from grant numberDMR-0097611, awarded by the National Science Foundation. The governmenthas certain rights in the invention.

INTRODUCTION

Dyes which, when irradiated with light of a wavelength absorbed by thesesubstances, emit light of a (usually) different wavelength are referredto as fluorescent dyes. Fluorescent dyes find us in a variety ofapplications in biochemistry, biology and medicine, e.g. in diagnostickits, in microscopy or in drug screening. Fluorescent dyes arecharacterized by a number of parameters allowing a user to select asuitable dye depending on the desired purpose. This includes theexcitation wavelength maximum, the emission wavelength maximum, theStokes shift, the extinction coefficient .epsilon, the fluorescencequantum yield, and the fluorescence lifetime. Dyes may be selectedaccording to the application of interest to, e.g., allow penetration ofexciting radiation into biological, to minimize background fluorescenceand to achieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a dye.

SUMMARY

Light harvesting luminescent multichromophores that are configured uponexcitation to transfer energy to, and amplify the emission from, anacceptor signaling chromophore in energy-receiving proximity therewithare provided. Also provided are compositions for labelling a target. Thelabelling composition may include a donor light harvestingmultichromophore and an acceptor signaling chromophore inenergy-receiving proximity to the donor light harvestingmultichromophore. Also provided is an aqueous composition for labellinga target, including: a donor light harvesting multichromophore; anacceptor signaling chromophore in energy-receiving proximity therewith;and a sensor biomolecule. Methods for using the subject compositions todetect a target are also provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 presents the emission spectra of a composition including asignaling chromophore (e.g. PNA-C*) that is either connected toconjugated polymer 1 (e.g., via binding of complementary DNA to PNA-C*)or not connected to polymer 1 (e.g., when non-complementary DNA fails tobind to PNA-C*), by excitation of polymer 1. PNA-C* and the respectiveDNA were added together in water at pH=5.5. The spectra are normalizedwith respect to the emission of polymer 1.

FIG. 2. Comparison of the intensity of signaling chromophore (e.g., EB(EB=Ethidium bromide)) emission from various compositions where aconjugated polymer and a signaling chromophore are connected via abiomolecule, e.g., polymer/ds-DNA/EB in 50 mmol phosphate buffer(pH=7.4) with [ds-DNA]=1.0 E⁸ M, [Polymer RU]=2.0 E⁷ M, [EB]=1.1 E⁶ M.Emission intensity was normalized relative to the E value at theexcitation wavelength.

DEFINITIONS

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention,

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a target polynucleotide” includes a plurality of targetpolynucleotides, reference to “a signaling chromophore” includes aplurality of such chromophores, reference to “a sensor PNA” includes aplurality of sensor PNAs, and the like. Additionally, use of specificplural references, such as “two,” “three,” etc., read on larger numbersof the same subject unless the context clearly dictates otherwise.

Terms such as “connected,” “attached,” “linked” and conjugated are usedinterchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each subrange between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed within the invention. Where a value beingdiscussed has inherent limits, for example where a component can bepresent at a concentration of from 0 to 100%, or where the pH of anaqueous solution can range from 1 to 14, those inherent limits arespecifically disclosed. Where a value is explicitly recited, it is to beunderstood that values which are about the same quantity or amount asthe recited value are also within the scope of the invention, as areranges based thereon. Where a combination is disclosed, eachsubcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention.

Conversely, where different elements or groups of elements aredisclosed, combinations thereof are also disclosed. Where any element ofan invention is disclosed as having a plurality of alternatives,examples of that invention in which each alternative is excluded singlyor in any combination with the other alternatives are also herebydisclosed; more than one element of an invention can have suchexclusions, and all combinations of elements having such exclusions arehereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. These terms refer only to the primary structure of themolecule. Thus, the terms include triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation and/or by capping, and unmodified forms of thepolynucleotide. “Complementary” or “substantially complementary” refersto the ability of a first specific binding member to specifically bindto a second specific binding member (e.g., hybridize or base pairbetween nucleotides or nucleic acids, such as, for instance, between asensor peptide nucleic acid and a target polynucleotide). Complementarynucleotides are, generally, A and T (or A and U), or C and G. Twosingle-stranded polynucleotides or PNAs are said to be substantiallycomplementary when the bases of one strand, optimally aligned andcompared and with appropriate insertions or deletions, pair with atleast about 80% of the bases of the other strand, usually at least about90% to 95%, and more preferably from about 98 to 100%.

Alternatively, substantial complementarity exists when a polynucleotideor PNA will hybridize under selective hybridization conditions to itscomplement, Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25bases, preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984).

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one specific binding member (e.g., apolynucleotide or PNA) to bind to its complementary specific bindingmember in a sample as compared to another noncomplementary component inthe sample.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of amino acids linked through peptide bonds. The termsdo not refer to a specific length of the product. Thus, “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The terms include polypeptides containing modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations, and sulphations. In addition, protein fragments,analogs (including amino acids not encoded by the genetic code, e.g.homocysteine, ornithine, D-amino acids, and creatine), natural orartificial mutants or variants or combinations thereof, fusion proteins,and proteins comprising derivatized residues (e.g. alkylation of aminegroups, acetylations or others esterifications of carboxyl groups) andthe like are included within the meaning of polypeptide.

“Multiplexing” herein refers to an assay or other analytical method inwhich multiple analytes can be assayed simultaneously.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs singly or multiply andinstances in which it does not occur at all. For example, the phrase“optionally substituted alkyl” means an alkyl moiety that may or may notbe substituted and the description includes both unsubstituted,monosubstituted, and polysubstituted alkyls.

“Alkyl” refers to a branched, unbranched or cyclic saturated hydrocarbongroup of 1 to 24 carbon atoms optionally substituted at one or morepositions, and includes polycyclic compounds. Examples of alkyl groupsinclude optionally substituted methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like, as well as cycloalkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and norbornyl. The term “lower alkyl” refers toan alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.Exemplary substituents on substituted alkyl groups include hydroxyl,cyano, alkoxy, ═O, ═S, —NO₂, halogen, haloalkyl, heteroalkyl,carboxyalkyl, amine, amide, thioether and —SH.

“Alkoxy” refers to an “—Oalkyl” group, where alkyl is as defined above.A “lower alkoxy” group intends an alkoxy group containing one to six,more preferably one to four, carbon atoms.

“Alkenyl” refers to a branched, unbranched or cyclic hydrocarbon groupof 2 to 24 carbon atoms containing at least one carbon-carbon doublebond optionally substituted at one or more positions. Examples ofalkenyl groups include ethenyl, 1-propenyl, 2-propenyl(allyl),1-methylvinyl, cyclopropenyl, 1-butenyl, 2-butenyl, isobutenyl,1,4-butadienyl, cyclobutenyl, 1-methylbut-2-enyl, 2-methylbut-2-en-4-yl,prenyl, pent-1-enyl, pent-3-enyl, 1,1-dimethylallyl, cyclopentenyl,hex-2-enyl, 1-methyl-1-ethylallyl, cyclohexenyl, heptenyl,cycloheptenyl, octenyl, cyclooctenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like. Preferred alkenylgroups herein contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms, preferably 2 to 4carbon atoms. The term “cycloalkenyl” intends a cyclic alkenyl group of3 to 8, preferably 5 or 6, carbon atoms. Exemplary substituents onsubstituted alkenyl groups include hydroxyl, cyano, alkoxy, ═O, ═S,—NO₂, halogen, haloalkyl, heteroalkyl, amine, thioether and —SH.

“Alkenyloxy” refers to an “—Oalkenyl” group, wherein alkenyl is asdefined above.

“Alkylaryl” refers to an alkyl group that is covalently joined to anaryl group. Preferably, the alkyl is a lower alkyl. Exemplary alkylarylgroups include benzyl, phenethyl, phenopropyl, 1-benzylethyl,phenobutyl, 2-benzylpropyl and the like.

“Alkylaryloxy” refers to an “—Oalkylaryl” group, where alkylaryl is asdefined above.

“Alkynyl” refers to a branched or unbranched hydrocarbon group of 2 to24 carbon atoms containing at least one —C≡O— triple bond, optionallysubstituted at one or more positions. Examples of alkynyl groups includeethynyl, n-propynyl, isopropynyl, propargyl, but-2-ynyl,3-methylbut-1-ynyl, octynyl, decynyl and the like. Preferred alkynylgroups herein contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6, preferably 2 to 4, carbon atoms, andone —C≡O— triple bond. Exemplary substituents on substituted alkynylgroups include hydroxyl, cyano, alkoxy, ═O, ═S, —NO2, halogen,haloalkyl, heteroalkyl, amine, thioether and —SH.

“Amide” refers to —C(O)NR′R″, where R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and alkylaryl.

“Amine” refers to an —N(R′)R″ group, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Aryl” refers to an aromatic group that has at least one ring having aconjugated pi electron system and includes carbocyclic, heterocyclic,bridged and/or polycyclic aryl groups, and can be optionally substitutedat one or more positions. Typical aryl groups contain 1 to 5 aromaticrings, which may be fused and/or linked. Exemplary aryl groups includephenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, biphenyl, indenyl, benzofuranyl, indolyl, naphthyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl,pyrrolopyridinyl, purinyl, tetralinyl and the like. Exemplarysubstituents on optionally substituted aryl groups include alkyl,alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl,aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturatedoptionally substituted rings, halogen, haloalkyl, heteroalkyl, —S(O)R,sulfonyl, —SO₃R, —SR, —NO₂, —NRR′, —OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R,—(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′ where n is 0-4, and wherein R and R′are independently H, alkyl, aryl or alkylaryl.

“Aryloxy” refers to an “—Oaryl” group, where aryl is as defined above.

“Carbocyclic” refers to an optionally substituted compound containing atleast one ring and wherein all ring atoms are carbon, and can besaturated or unsaturated.

“Carbocyclic aryl” refers to an optionally substituted aryl groupwherein the ring atoms are carbon.

“Halo” or “halogen” refers to fluoro, chloro, bromo or iodo. “Halide”refers to the anionic form of the halogens.

“Haloalkyl” refers to an alkyl group substituted at one or morepositions with a halogen, and includes alkyl groups substituted withonly one type of halogen atom as well as alkyl groups substituted with amixture of different types of halogen atoms. Exemplary haloalkyl groupsinclude trihalomethyl groups, for example trifluoromethyl.

“Heteroalkyl” refers to an alkyl group wherein one or more carbon atomsand associated hydrogen atom(s) are replaced by an optionallysubstituted heteroatom, and includes alkyl groups substituted with onlyone type of heteroatom as well as alkyl groups substituted with amixture of different types of heteroatoms. Heteroatoms include oxygen,sulfur, and nitrogen. As used herein, nitrogen heteroatoms and sulfurheteroatoms include any oxidized form of nitrogen and sulfur, and anyform of nitrogen having four covalent bonds including protonated forms.An optionally substituted heteroatom refers to replacement of one ormore hydrogens attached to a nitrogen atom with alkyl, aryl, alkylarylor hydroxyl.

“Heterocyclic” refers to a compound containing at least one saturated orunsaturated ring having at least one heteroatom and optionallysubstituted at one or more positions. Typical heterocyclic groupscontain 1 to 5 rings, which may be fused and/or linked, where the ringseach contain five or six atoms. Examples of heterocyclic groups includepiperidinyl, morpholinyl and pyrrolidinyl. Exemplary substituents foroptionally substituted heterocyclic groups are as for alkyl and aryl atring carbons and as for heteroalkyl at heteroatoms.

“Heterocyclic aryl” refers to an aryl group having at least 1 heteroatomin at least one aromatic ring. Exemplary heterocyclic aryl groupsinclude furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-loweralkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl,tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl,phenanthrolinyl, purinyl and the like.

“Hydrocarbyl” refers to hydrocarbyl substituents containing 1 to about20 carbon atoms, including branched, unbranched and cyclic species aswell as saturated and unsaturated species, for example alkyl groups,alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, andthe like. The term “lower hydrocarbyl” intends a hydrocarbyl group ofone to six carbon atoms, preferably one to four carbon atoms.

A “substituent” refers to a group that replaces one or more hydrogensattached to a carbon or nitrogen. Exemplary substituents include alkyl,alkylidenyl, alkylcarboxy, alkoxy, alkenyl, alkenylcarboxy, alkenyloxy,aryl, aryloxy, alkylaryl, alkylaryloxy, —OH, amide, carboxamide,carboxy, sulfonyl, ═O, ═S, —NO₂, halogen, haloalkyl, fused saturated orunsaturated optionally substituted rings, —S(O)R, —SO₃R, —SR, —NRR′,—OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R, —(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′where n is 0-4, and wherein R and R′ are independently H, alkyl, aryl oralkylaryl. Substituents also include replacement of a carbon atom andone or more associated hydrogen atoms with an optionally substitutedheteroatom.

“Sulfonyl” refers to —S(O)₂R, where R is alkyl, aryl, —C(CN)═C-aryl,—CH2CN, alkylaryl, or amine.

“Thioamide” refers to —C(S)NR′R″, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Thioether” refers to —SR, where R is alkyl, aryl, or alkylaryl.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as: hybrid(chimeric) antibody molecules (see, for example, Winter et al. (1991)Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 and F(ab)fragments; Fv molecules (noncovalent heterodimers, see, for example,Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich etal. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see,for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883);dimeric and trimeric antibody fragment constructs; minibodies (see,e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) JImmunology 149B:120-126); humanized antibody molecules (see, forexample, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al.(1988) Science 239:1534-1536; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994); and, any functional fragmentsobtained from such molecules, wherein such fragments retainspecific-binding properties of the parent antibody molecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. Thus, the termencompasses antibodies obtained from murine hybridomas, as well as humanmonoclonal antibodies obtained using human hybridomas or from murinehybridomas made from mice expression human immunoglobulin chain genes orportions thereof. See, e.g., Cote, et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, 1985, p. 77.

DETAILED DESCRIPTION

As summarized above, compositions including luminescent light harvestingmultichromophores that are configured upon excitation to transfer energyto, and amplify the emission from, an acceptor signaling chromophore inenergy-receiving proximity therewith are provided. The light harvestingmultichromophores find use in a variety of applications, includinganalyte detection applications.

Light Harvesting, Luminescent Multichromophore Systems

Luminescent light harvesting multichromophore systems are efficientlight absorbers by virtue of the multiple chromophores they include.Light harvesting multichromophores of interest include, but are notlimited to, conjugated polymers, aggregates of conjugated molecules,luminescent dyes attached via side chains to saturated polymers,semiconductor quantum dots and dendritic structures. In some instances,the light harvesting multichromophore system includes a light harvestingconjugated polymer. For example, each repeat unit on a conjugatedpolymer can be considered as a contributing chromophore, quantum dotsare made up of many atoms, a saturated polymer can be functionalizedwith many luminescent dye molecules on side chains, and dendrimers canbe synthesized containing many covalently bonded individualchromophores. Attachment of chromophore assemblies onto solid supports,such as polymer beads or surfaces, can also be used for lightharvesting.

In some instances, the water soluble, light harvesting multichromophorehas a luminescent emission spectrum, i.e., the multichromophore isitself luminescent. In some cases, water soluble, light harvestingmultichromophore has high luminescence quantum efficiency. In certaincases, the water soluble, light harvesting multichromophore has highfluorescent efficiency. Light harvesting multichromophore systems canalso efficiently transfer energy to nearby luminescent species (e.g.,“signaling chromophores”). Mechanisms for energy transfer include, forexample, resonant energy transfer (Förster (or fluorescence) resonanceenergy transfer, FRET), quantum charge exchange (Dexter energy transfer)and the like. In some cases, however, these energy transfer mechanismsare relatively short range; that is, close proximity of the lightharvesting multichromophore system to the signaling chromophore isrequired for efficient energy transfer. As used herein, the term “energyreceiving proximity” refers to an arrangement of the light harvestingmultichromophore system and the signaling chromophore sufficient forefficient energy transfer, e.g., via FRET. Under conditions forefficient energy transfer, amplification of the emission from thesignaling chromophore occurs when the number of individual chromophoresin the light harvesting multichromophore system is large; that is, theemission from the signaling chromophore is more intense when theincident light (the “pump light”) is at a wavelength which is absorbedby the light harvesting multichromophore system than when the signalingchromophore is directly excited by the pump light.

When the average distance between the light harvesting multichromophoreand the signaling chromophore is too large for effective energytransfer, there is little or no emission from the signaling chromophore.As such, the light harvesting multichromophore may be configured uponexcitation to transfer energy to, and amplify the emission from, anacceptor signaling chromophore in energy-receiving proximity therewith,

The light harvesting multichromophore and the signaling chromophore (C*)are selected so that the absorption bands of the two chromophores haveminimal overlap and so that the luminescent emission spectra of the twospecies are at different wavelengths. In some instances, the watersoluble, light harvesting multichromophore has a luminescent emissionspectrum, i.e., the multichromophore is luminescent.

As shown by Förster, dipole-dipole interactions lead to long-rangeresonance energy transfer (FRET) from a donor chromophore to an acceptorchromophore. The energy transfer efficiency (E) is proportional to 1/r⁶,where r is the donor-acceptor distance, and the overlap integral, asshown in Equation 1.

$\begin{matrix}{E \propto {{\frac{1}{r^{6}} \cdot \underset{0}{\overset{\infty}{o}}}{F_{D}(\lambda)}{ɛ_{A}(\lambda)}\lambda^{4}{d\lambda}}} & (1)\end{matrix}$

The distance requirement for energy transfer in the compositionsdescribed herein may be controlled by the configuration of the donor andthe acceptor. The overlap integral expresses the spectral overlapbetween the emission of the donor and the absorption of the acceptor.The components of the labelling composition and their relativeconfiguration can be selected so that their optical properties meet thisrequirement.

In some instances, the light harvesting multichromophores describedherein are soluble in aqueous solutions and other polar solvents, and insome cases are soluble in water, i.e., they are water-soluble. By“water-soluble” is meant that the material exhibits solubility in apredominantly aqueous solution, which, although comprising more than 50%by volume of water, does not exclude other substances from thatsolution, including without limitation buffers, blocking agents,co-solvents, salts, metal ions and detergents. It is understood that thelight harvesting multichromophores may be selected and/or adapted toachieve a variety of desirable characteristics (such as watersolubility) using any convenient methods and materials, such as themethods and materials described by N. Angelova and D. Hunkeler in“Rationalizing the design of polymeric biomaterials”, TIBTECH, October1999, 17, 409421; and S. Zalipsky in “Functionalized poly(ethyleneglycol) for preparation of biologically relevant conjugates”,Bioconjugate Chemistry 1995, 6 (2), 150-165. In some instances, thelight harvesting multichromophores include water-soluble groups whichimprove the water solubility of the molecules. In certain instances, thelight harvesting multichromophore is conjugated to another water solublemolecule, such as a biomolecule. In certain cases, the light harvestingmultichromophore is conjugated to a sensor.

Any convenient water-soluble groups (WSGs) may be utilized in thesubject light harvesting multichromophores. The term “water-solublegroup” refers to a functional group that is well solvated in aqueousenvironments and that imparts improved water solubility to the moleculesto which it is attached. In some embodiments, a WSG increases thesolubility of the multichromophore in a predominantly aqueous solution(e.g., as described herein), as compared to a multichromophore whichlacks the WSG. The water soluble groups may be any convenienthydrophilic group that is well solvated in aqueous environments. In somecases, the hydrophilic water soluble group is charged, e.g., positivelyor negatively charged. In certain cases, the hydrophilic water solublegroup is a neutral hydrophilic group. In some embodiments, the WSG is ahydrophilic polymer, e.g., a polyethylene glycol, a cellulose, achitosan, or a derivative thereof.

As used herein, the terms “polyethylene oxide”, “PEO”, “polyethyleneglycol” and “PEG” are used interchangeably and refer to a polymerincluding a chain described by the formula —(CH₂—CH₂—O—)_(n)— or aderivative thereof. In some embodiments, “n” is 5000 or less, such as1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 orless, 30 or less, 20 or less, 15 or less, such as 5 to 15, or 10 to 15.It is understood that the PEG polymer may be of any convenient lengthand may include a variety of terminal groups, including but not limitedto, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminalgroups. Functionalized PEGs that may be adapted for use in the subjectmultichromophores include those PEGs described by S. Zalipsky in“Functionalized poly(ethylene glycol) for preparation of biologicallyrelevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165.

Water soluble groups of interest include, but are not limited to,carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate,ester, polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine,ammonium, guanidinium, polyamine and sulfonium, polyalcohols, straightchain or cyclic saccharides, primary, secondary, tertiary, or quaternaryamines and polyamines, phosphonate groups, phosphinate groups, ascorbategroups, glycols, including, polyethers, —COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺,Y′, (CH₂CH₂O)_(p)R and mixtures thereof, where Y′ can be any halogen,sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, eachR can be independently H or an alkyl (such as methyl) and M′ can be acationic counterion or hydrogen, —(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy),—(CH₂CH₂O)_(yy)CH₂CH₂X—, —X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, andpolyethylene glycol, wherein yy is selected from 1 to 1000, X isselected from O, S, and NR^(ZZ), and R^(ZZ) and R′ are independentlyselected from H and C1-3 alkyl.

In some embodiments, the light harvesting multichromophore includescationic WSGs and is polycationic. Any suitable cationic WSGs may beincorporated into the light harvesting multichromophores, including, butnot limited to, ammonium groups, guanidinium groups, histidines,polyamines, pyridinium groups, and sulfonium groups.

The water solubility of light harvesting multichromophores (e.g., CPs),finds use in detecting biological targets, and in some cases is achievedby including water-soluble groups (e.g., hydrophilic groups, such as PEGor modified PEG groups or charged groups attached to the CP backbone).In some instances, the light harvesting multichromophores are conjugatedpolymers, which include sulfonate or carboxylate functionalities.

Exemplary multichromophores which can be used include, but are notlimited to, conjugated polymers (which includes oligomers), saturatedpolymers or dendrimers incorporating multiple chromophores in any viablemanner, and semiconductor nanocrystals (SCNCs). In some cases, theconjugated polymers, saturated polymers and dendrimers can be preparedto incorporate multiple WSGs or can be derivatized after synthesis. Forexample, in Example 4, Schemes 2 and 3 depict the preparation ofconjugated polymers that may be adapted to include any convenientsubstituted alkyl (e.g., bromo-substituted alkyl) substituents. Anyconvenient substituents (such as water soluble groups) may be selectedfor inclusion in the subject conjugated polymers via polymerderivatization after polymer synthesis, e.g., via nucleophilicsubstitution of the bromoalkyl substituent groups with any convenientnucleophilic groups to produce heteroalkyl substituted conjugatedpolymers.

In some embodiments, a water soluble light harvesting multichromophoreis a conjugated polymer that includes water soluble groups. For example,water soluble conjugated molecules of interest which may be preparedaccording to the methods described herein include, but are not limitedto, polymer 1 (where n=2-100,000), oligomer 1 and oligomer 2, shownbelow. In some instances, the water soluble light harvestingmultichromophores have a structure selected from the formula of polymer1, the formula of oligomer 1 or the formula of oligomer 2, depictedbelow. However, the specific molecular structures of polymer 1, oligomer1 and oligomer 2, depicted below are not critical, and any water solublelight harvesting molecules can be used.

A specific example is shown in structure 1 where the water solubleconjugated polymer ispoly((9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene)(denoted in the following as polymer 1). In some cases, the particularsize of the subject conjugated polymers is not critical, so long as theCP is able to absorb light and transfer energy to signaling chromophoresthat are configured in energy receiving proximity. In some cases, valuesof “n” fall within the range of two to about 100,000. This specificmolecular structure is not critical; any water soluble conjugatedpolymer with relatively high luminescence quantum efficiency can beused.

In some instances, the water soluble light harvesting multichromophoreis a conjugated oligomer, such as a water soluble, cationic, luminescentconjugated oligomer, as shown below (denoted herein as oligomer 2):

In certain cases, in aqueous media, oligomers such as 2 may be moresoluble than their polymeric counterparts, and hydrophobic interactionswith sensor biomolecules may be less important for 2 than for largerpolymer structures.

Addition of organic solvents to the subject compositions, for example awater miscible organic solvent such as ethanol, can result in a decreasein background (C*) emission. The presence of the organic solvent candecrease hydrophobic interactions between the light harvestingmultichromophore and another component of the assay and thereby reducebackground signal.

Conjugated Polymers

In some embodiments, the light harvesting multichromophore is aconjugated polymer. Conjugated polymers (CPs) are characterized by adelocalized electronic structure and, in some cases, can be used ashighly responsive optical reporters for chemical and biological targets.In a CP, the effective conjugation length may be substantially shorterthan the length of the polymer chain, and thus the backbone may containa large number of conjugated segments in close proximity. In someinstances, conjugated polymers are efficient for light harvesting andprovide for optical amplification via Förster energy transfer to asignalling chromophore. In certain instances, water-soluble CPs showexceptional fluorescence quenching efficiencies in the presence ofacceptor in energy-receiving proximity and are of interest fortransduction of biological recognition events, among other uses.

In one embodiment, a conjugated polymer is represented by Formula A:

where:

CP₁, CP₂, CP₃, and CP₄ are optionally substituted conjugated polymersegments or oligomeric structures, and may be the same or different fromone another;

LU₁ and LU₂ are each independently a linker unit;

G₁ and G are each independently a capping unit;

a, b, c, d, e and f are each independently 0 to 250; and

m and n are each independently 0 to 10,000, where m+n>1.

CP₁, CP₂, CP₃, and CP₄ may be independently aromatic repeat units, and,in some cases, may be selected from the group consisting of benzene,naphthalene, anthracene, fluorene, thiophene, furan, pyridine, andoxadiazole, each optionally substituted.

In some embodiments, the formula contains linker units LU₁ and LU₂ whichmay be angled linkers (e.g., as described herein) and, in some cases,can be mono- or polycyclic optionally substituted aryl groups having 5to 20 atoms (e.g., an aromatic repeat unit of Table 1 or 2). The linkerunits may be evenly or randomly distributed along the polymer mainchain. Aromatic rings of interest include those which also produce aspatial twist of the polymer main chain, preventing the conjugatedpolymer from forming a plane across that linker unit.

In some embodiments, LU₁ and LU₂ are independently selected from thegroup consisting of benzene derivatives incorporated into the polymer inthe 1,2 or 1,3-positions; naphthalene derivatives incorporated into thepolymer in the 1,2-, 1,3-, 1,6-, 1,7-, 1,8-positions; anthracenederivatives incorporated into the polymer in the 1,2-, 1,3-, 1,6-, 1,7-,1,8-, and 1,9-positions; biphenyl derivatives incorporated into thepolymer in the 2,3-, 2,4-, 2,6-, 3,3′-, 3,4-, 3,5-, 2,2′-, 2,3′-, 2,4′-,and 3,4′-positions; and corresponding heterocycles. The position numbersare given with reference to unsubstituted carbon-based rings, but thesame relative positions of incorporation in the polymer are encompassedin substituted rings and/or heterocycles should their distribution ofsubstituents change the ring numbering.

In some instances, CP₁, CP₂, CP₃, CP₄, LU₁ and LU₂ are each optionallysubstituted at one or more positions with one or more groups selectedfrom —R¹-A, —R²-B, —R³-C, —R⁴-D and —R⁵-I, which may be attached throughbridging functional groups -E- and -F- (see e.g., the units depicted inTables 1 and 2). In some instances, R¹, R², R³, R⁴ and R⁵ areindependently selected from alkyl, alkenyl, alkoxy, alkynyl, and aryl,alkylaryl, arylalkyl, and polyalkylene oxide, each optionallysubstituted, which may contain one or more heteroatoms, or may be notpresent. In some instances, R¹, R², R³, R⁴ and R⁵ can be independentlyselected from C₁₋₂₂ alkyl, C₁₋₂₂ alkoxy, C₁₋₂₂ ester, polyalkylene oxidehaving from 1 to about 22 carbon atoms, cyclic crown ether having from 1to about 22 carbon atoms, or not present. In some instances, R¹, R², R³,R⁴ and R⁵ may be selected from straight or branched alkyl groups having1 to about 12 carbon atoms, or alkoxy groups with 1 to about 12 carbonatoms. It is to be understood that more than one functional group may beappended to the rings as indicated in the formulas at one or morepositions.

In some instances, A, B, C, D and I are independently selected from H, aWSG, —SiR′R″R′″, —N⁺R′R″R′″, a guanidinium group, histidine, apolyamine, a pyridinium group, and a sulfonium group. In some instances,R′, R″ and R′″ are independently selected from the group consisting ofhydrogen, C₁₋₁₂ alkyl and C₁₋₁₂ alkoxy and C₃₋₁₀ cycloalkyl. In certainembodiments, R′, R″ and R′″ are lower alkyl or lower alkoxy groups. Insome embodiments, E and F are independently selected from not present,—O—, —S—, —C(O)—, —C(O)O—, —C(R)(R′)—, —N(R′)—, and —Si(R′)(R″), whereinR′ and R″ are as defined above. In some embodiments, E and F are —O—. Insome embodiments, E and F are not present. In some instances, A, B, C, Dand I are H. In certain instances, A, B, C, D and I are independently aWSG.

In some cases, X is O, S, Se, —N(R′)— or —C(R′)(R″)—, and Y and Z areindependently selected from —C(R)═ and —N═, where R, R′ and R″ are asdefined above.

In certain instances, m and n are independently 0 to 10,000, whereinm+n>1. In certain embodiments, m and n are each independently 0 to 20and in some cases from 0 to 10. Each repeat of m and n may be the sameas or different than the other repeats thereof. In some embodiments, band e are independently 0 to 250, where b+e>1. In certain cases, a, c, dand f are independently 0 to 250. In some embodiments, b and e are each0.

Any convenient capping units may be utilized in the conjugated polymers.In some instances, G and G₁ are capping units and may be the same ordifferent. The capping units may be activated units that allow furtherchemical reaction to the terminal of the polymer chain. In some cases, Gand G₁ may be a group that includes a reactive functional group suitablefor conjugation to a biomolecule. Any convenient activated units may beutilized as capping units G and/or G₁ in the subject CPs. The cappingunits may be nonactivated termination units. In certain instances, G andG₁ can be independently selected from hydrogen, optionally substitutedaryl, halogen substituted aryl, boronic acid substituted aryl, andboronate radical substituted aryl.

Also provided are conjugated polymer compositions of formula A whereCP₁, CP₂, CP₃, CP₄, LU₁ and LU₂ are each optionally substituted at oneor more positions with one or more groups selected from —R¹-A, —R²-B,—R³-C, —R⁴-D and —R⁵-I where A, B, C, D and I are each H, which may beattached via bridging functional groups -E- and -F-. In some instances,R¹, R², R³, R⁴ and R⁵ are independently selected from alkyl, alkenyl,alkoxy, alkynyl, and aryl, alkylaryl, arylalkyl, and polyalkylene oxide,each optionally substituted, which may contain one or more heteroatoms,or may be not present. In some embodiments, E and F are independentlyselected from not present, —O—, —S—, —C(O)—, —C(O)O—, —C(R)(R′)—,—N(R′)—, and —Si(R′)(R″), wherein R′ and R″ are as defined above.

In certain embodiments of Formula A, at least one of —R¹-A, —R²-B,—R³-C, —R⁴-D and —R⁵-I includes a water soluble group (WSG). In someinstances, R³-C and —R⁴-D each independently include a water solublegroup. In certain embodiments, —R¹-A, —R²-B and —R⁵-I are each H. Incertain instances, E and R⁵-I are not present. In certain embodiments,R³ and R⁴ are alkyl, each optionally substituted.

In certain embodiments the light harvesting multichromophore is aconjugated polymer including a conjugated segment having the structure:

where n, E, —R¹-A, —R²-B, —R³-C, —R⁴-D and —R⁵-I are as defined above.In certain embodiments, at least one of —R¹-A, —R²-B, —R³-C, —R⁴-D and—R⁵-I includes a water soluble group (WSG). In some instances, R³-C and—R⁴-D each independently include a water soluble group. In certainembodiments, —R¹-A, —R²-B and —R⁵-I are each H. In certain instances, Eand R⁵-I are not present. In certain embodiments, R³ and R⁴ are alkyl,each optionally substituted.

Aromatic repeat units of interest are shown in Table 1 below, andrepresentative polymeric segments and oligomeric structures are shown inTable 2.

TABLE 1 Aromatic repeat units of interest for the construction ofconjugated segments and oligomeric structures.

TABLE 2 Examples of conjugated segments and oligomeric structures of CPs

Conformationally Flexible CPs

Conjugated polymers (CPs) are efficient light-gathering molecules withproperties desirable for a variety of applications. Conjugated polymerscan serve as light harvesting materials and signal transducers influorescent biosensor applications. These molecules can detect,transduce and/or amplify chemical, biological or physical informationinto optical and/or electrical signals. CPs can provide the advantage ofcollective response relative to non-interacting small molecules. Thiscollective response influences optoelectronic properties, such asFörster resonance energy transfer (FRET), electrical conductivity andfluorescence efficiency, properties which can be used to report, or“transduce,” target analyte presence.

Aspects of the invention include conjugated polymers (CPs) includingmonomers which perturb the polymer's ability to form rigid-rodstructures, allowing them to form a greater range of three-dimensionalstructures. The monomers may include aromatic molecules havingattachment points to the adjacent subunits of the polymer which form anangle of greater than about 25°. The monomers may introduce a torsionaltwist in the conjugated polymer, thereby further disrupting the abilityof the polymer to form a rigid-rod structure.

A synthetic method is also provided for producing CPs with a range ofbackbone regiochemistries. Such CPs exhibit facile energy transferamongst polymer segments which results in similar emission propertiesand FRET function. In some instances, the flexible CPs are efficientexcitation donors with respect to particular biomolecules and find usein bioassays that take advantage of the optical amplification ofwater-soluble conjugated polymers.

In one aspect, a plurality of CPs with different structures areprovided, which may be in the form of a library. The CPs may be testedfor any property of interest. For example, the CPs may be tested forincreased fluorescent efficiency, for decreased self-quenching,increased Stoke's shift, and for emission wavelength.

In some embodiments, modification of polymer shape is achieved throughfractional incorporation of meta and para linkages on phenylene unitsadjacent to fluorenyl monomer units. The meta to para ratio may becontrolled during the polymerization reaction by use of, for example,1,3-phenylenebisboronic acid and 1,4-phenylenebisboronic acid inappropriate ratios. The corresponding polymers may have ratios of metato para linkages ranging from 0 to 100%. In some cases, the introductionof the meta linkage not only permits shape control, but also providesthe possibility of energy transfer along the polymer main chain, orbetween different polymer segments, since fragments containing a higherfraction of para linkages may be of lower energy level, and may behaveas low energy traps.

Conformationally flexible conjugated polymers (CPs) may include angledlinkers with a substitution pattern (or regiochemistry) capable ofperturbing the polymers' ability to form rigid-rod structures, allowingthe CPs to have a greater range of three-dimensional structures. The CPsmay include at least three subunits with at least one angled linker,which may be internal and/or an end unit, and may comprise at least 4,5, 6, 8, 10, 15, 20, 25 or more subunits. The CPs may include up toabout 100, 200, 300, 500, 1000, 2000, 5000, 10000, 20000, 50000 or moresubunits.

The angled linker(s) are optionally substituted aromatic moleculeshaving at least two separate bonds to other polymer components (e.g.,monomers, block polymers, end groups) that are capable of forming anglesrelative to one another which disrupt the overall ability of the polymerto form an extended rigid-rod structure (although significant regionsexhibiting such structure may remain.) The angled linkers may bebivalent or polyvalent.

The angle which the angled linkers are capable of imparting to thepolymeric structure is determined as follows. Where the two bonds toother polymeric components are coplanar, the angle can be determined byextending lines representing those bonds to the point at which theyintersect, and then measuring the angle between them. Where the twobonds to other polymeric components are not coplanar, the angle can bedetermined as follows: a first line is drawn between the two ring atomsto which the bonds attach; two bond lines are drawn, one extending fromeach ring atom in the direction of its respective bond to the otherpolymeric component to which it is joined; the angle between each bondline and the first line is fixed; and the two ring atoms are then mergedinto a single point by shrinking the first line to a zero length; theangle then resulting between the two bond lines is the angle the angledlinker imparts to the CP.

The angle which an angled linker is capable of imparting to the polymeris in some cases less than 155°, and may be less than 150°, less than145°, less than 140°, less than 135°, less than 130°, less than 125°,less than 120°, less than 115°, less than 110°, less than 105°, lessthan 100°, less than 95°, less than 90°, less than 85°, less than 80°,less than 75°, less than 70°, less than 65°, less than 60°, less than55°, or less than 50°. The angled linker may form an angle to itsadjacent polymeric units of about 25°, 30°, 35°, 40°, 45°, 50°, 60° ormore. The angled linker may introduce a torsional twist in theconjugated polymer, thereby further disrupting the ability of thepolymer to form a rigid-rod structure. For angled linkers having aninternally rotatable bond, such as polysubstituted biphenyl, the angledlinker may be capable of imparting an angle of less than about 155° inat least one orientation.

For six-membered rings, such angles can be achieved through ortho ormeta linkages to the rest of the polymer. For five-membered rings,adjacent linkages fall within this range. For eight-membered rings,linkages extending from adjacent ring atoms, from alternate ring atoms(separated by one ring atom), and from ring atoms separated by two otherring atoms fall within this range. Ring systems with more than eightring atoms may be used. For polycyclic structures, even more diverselinkage angles can be achieved.

Exemplary linking units which meet these limitations include benzenederivatives incorporated into the polymer in the 1, 2 or 1,3-positions;naphthalene derivatives incorporated into the polymer in the 1,2-, 1,3-,1,6-, 1,7-, 1,8-positions; anthracene derivatives incorporated into thepolymer in the 1,2-, 1,3-, 1,6-, 1,7-, 1,8-, and 1,9-positions; biphenylderivatives incorporated into the polymer in the 2,3-, 2,4-, 2,6-,3,3′-, 3,4-, 3,5-, 2,2′-, 2,3′-, 2,4′-, and 3,4′-positions; andcorresponding heterocycles. The position numbers are given withreference to unsubstituted carbon-based rings, but the same relativepositions of incorporation in the polymer are encompassed in substitutedrings and/or heterocycles should their distribution of substituentschange the ring numbering.

The CP in some cases contains at least 0.01 mol % of the angled linker,and may contain at least 0.02 mol %, at least 0.05 mol %, at least 0.1mol %, at least 0.2 mol %, at least 0.5 mol %, at least 1 mol %, atleast 2 mol %, at least 5 mol %, at least 10 mol %, at least 20 mol %,or at least 30 mol %. The CCP may contain up to 100 mol % of the angledlinker, and may contain 99 mol % or less, 90 mol % or less, 80 mol % orless, 70 mol % or less, 60 mol % or less, 50 mol % or less, or 40 mol %or less.

The CP can be a copolymer, and may be a block copolymer, a graftcopolymer, or both. The angled linker may be incorporated into the CPrandomly, alternately, periodically and/or in blocks. In one aspect, theangled linker can be selected from aromatic or heteroaromatic structuresin which the shortest link between the linking points to the polymerinvolves an even number of atoms bonded to one another.

In some instances, the conformationally flexible CPs are water soluble,and any or all of the subunits of the polymer may comprise one or morewater soluble groups, including the angled linker(s).

Synthesis of Conjugated Polymers

A synthetic approach of interest is as follows. A neutral conjugatedpolymer is formed by the Suzuki coupling of a targeted ratio of monomerunits, to produce a neutral conjugated polymer that may includeheteroalkyl substituents. For example, as depicted in example, Schemes 2and 3, 1,3-phenylenebisboronic acid and/or 1,4-phenylenebisboronic acidmay be coupled with 2,7-dibromo-9,9-bis(6′-bromohexyl)fluorene toproduce a substituted neutral CP. In some embodiments, the heteroalkylsubstituted conjugated polymer may be further derivatized with anyconvenient group. In some instances, a water-soluble conjugated polymeris produced by inclusion of a water soluble group in a monomeric unitduring polymer synthesis. In certain instances, a water soluble group isincluded via derivatization of a CP after polymer synthesis. Forexample, a water soluble group may be added via derivatization of a6′-bromohexyl substituted CP.

The Signaling Chromophore

Chromophores useful in the inventions described herein include anysubstance which can absorb energy from a light harvestingmultichromophore, when it is configured in energy-receiving proximity tothe multichromophore and emit light. Chemical methods for attaching(e.g., directly or indirectly) a signaling chromophore to a sensormolecule and/or another assay component, such as a light harvestingmultichromophore, are known.

The chromophore may be a lumophore or a fluorophore. Fluorophores ofinterest include, but are not limited to, fluorescent dyes,semiconductor nanocrystals, lanthanide chelates, and green fluorescentprotein. Exemplary fluorescent dyes include, but are not limited to,fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, acarboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®,Cy5.5®, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-aProtein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 5581568,BODIPY® 5641570, BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650,BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugatesthereof, and combinations thereof. Exemplary lanthanide chelates includeeuropium chelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art and may find use as a signalling chromophore; methodsof producing and utilizing semiconductor nanocrystals are described in:PCT Publ. No. WO 99/26299 published May 27, 1999, inventors Bawendi etal.; U.S. Pat. No. 5,990,479 issued Nov. 23, 1999 to Weiss et al.; andBruchez et al., Science 281:2013, 1998. Semiconductor nanocrystals canbe obtained with very narrow emission bands with well-defined peakemission wavelengths, allowing for a large number of different SCNCs tobe used as signaling chromophores in the same assay, optionally incombination with other non-SCNC types of signaling chromophores. In somecases, the signaling chromophore is a polynucleotide-specific dye suchas an intercalating dye. Other dyes and fluorophores of interest aredescribed at www.probes.com (Molecular Probes, Inc.).

In some cases, the signaling chromophore is a fluroescent protein, suchas a green fluorescent protein (GFP). The term “green fluorescentprotein” refers to both native Aequorea green fluorescent protein andmutated versions that have been identified as exhibiting alteredfluorescence characteristics, including altered excitation and emissionmaxima, as well as excitation and emission spectra of different shapes(Delagrave, S. et al. (1995) Bio/Technology 13:151-154; Heim, R. et al.(1994) Proc. Natl. Acad. Sci. USA 91:12501-12504; Heim, R. et al. (1995)Nature 373:663-664). Delgrave et al. isolated mutants of cloned AequoreaVictoria GFP that had red-shifted excitation spectra. Bio/Technology13:151-154 (1995). Heim, R. et al. reported a mutant (Tyr66 to His)having a blue fluorescence (Proc. Natl. Acad. Sci. (1994) USA91:12501-12504).

For multiplexed assays, a plurality of different signaling chromophorescan be used with detectably different emission spectra. In someembodiments, a second signaling chromophore may be directly orindirectly attached to another of the assay components and/or to asubstrate. In certain instances, the second signaling chromophore isused to receive energy from the initial signaling chromophore. Incertain instances, this configuration can provide for significantadditional selectivity. Energy can then be transferred from the excitedlight harvesting multichromophore to the initial signaling chromophore,which subsequently transfers energy to the second signaling chromophore,in an overall format that provides for detection of the target. Thiscascade of signaling chromophores can, in principle, be extended to useany number of signaling chromophores with compatible absorption andemission profiles.

Sensor Molecules

In some instances, a sensor molecule is provided that is complementaryto the target to be detected. Any convenient sensor molecules that arecomplementary to a target may be utilized in the subject compositionsand methods. Sensor molecules of interest include biomolecules,including but not limited to, a peptide or protein, a polynucleotidesuch as DNA or RNA, and an antibody. In certain embodiments, the sensorbiomolecule is an antibody. In some cases, the sensor molecule isattached to a signaling chromophore. A variety of chemical methods forattaching a signaling chromophore to sensor are known in the art.Specific sensor structures, including structures conjugated tochromophores, can be custom-made using commercial sources or chemicallysynthesized.

In some embodiments, the sensor molecule is a sensor PNA that iscomplementary to a target polynucleotide to be assayed, and has apredetermined sequence. In certain embodiments, the sensor PNA can bebranched, multimeric or circular, but is in some cases linear, and cancontain nonnatural bases. The molecular structures of PNAs are wellknown. PNAs can be prepared with any desired sequence of bases. Chemicalmethods for attaching the signaling chromophore to the sensor PNA arewell known. Specific sensor PNA structures, including structuresconjugated to chromophores, can be custom-made using commercial sourcesor chemically synthesized.

In some instances, the sensor molecule is a sensor polynucleotide thatis complementary to a target polynucleotide to be assayed, and has apredetermined sequence. The sensor polynucleotide can be branched,multimeric or circular, but is in some cases linear, and can containnonnatural bases. The sensor polynucleotide can be prepared with anydesired sequence of bases. Chemical methods for attaching the signalingchromophore to the sensor polynucleotide are known in the art. Specificsensor polynucleotide structures, including structures conjugated tochromophores, can be custom-made using commercial sources or chemicallysynthesized.

In some cases, the sensor molecule is a peptide or protein, e.g., atarget binding protein. Any protein sensor molecule which canspecifically bind to a target polynucleotide of interest can be employedin the compositions and methods disclosed. In some embodiments, thetarget binding protein is an antibody that specifically binds a targetbiomolecule. In certain embodiments, the target binding protein is asensor polynucleotide binding protein (PBP) that specifically binds to atarget polynucleotide to be assayed.

Protein biomolecules that find use as a sensor molecule for specificallybinding a target biomolecule in the subject compositions and methods maybe prepared using any convenient methods. In some cases, the proteinsensor molecule is synthesized by the solid phase method and/or can bepurified by HPLC and/or characterized by MALDI-TOF mass spectrum andamino acid analysis. The chemical methods for attaching a signalingchromophore to a protein sensor molecule are known. A specific exampleis the labelling composition including Tat-C* with fluorescein at theN-terminus.

Non-limiting examples of target binding proteins include, but are notlimited to, transcription factors, splicing factors, poly(A) bindingproteins, chromatin components, viral proteins, proteins which detectviral infection, replication factors, and proteins involved in mitoticand/or meiotic cell division. Examples of specific sensor proteins whichcan be used include Tat which binds to the Rev Responsive Element ofhuman immunodeficiency virus (HIV), the matrix protein M1 which binds toType A influenza virus RNA, and hnRNP U protein which binds topre-ribosomal RNA.

Compositions

Aspects of the invention include compositions for detecting a targetthat include a signaling chromophore configured in energy-receivingproximity to a light harvesting multichromophore, such thatamplification of the emission from the signaling chromophore occurs. Thesubject compositions include light harvesting multichromophores that areconfigured so that they can interact with a signaling chromophore whichis in energy-receiving proximity therewith by virtue of any convenientconnection. In some embodiments, the connection is an indirectconnection via a sensor molecule. In some instances, the subjectcomposition is utilized as a detection reagent in an assay to directlylabel a target biomolecule.

The proximity between a signaling chromophore and a light harvesting andluminescent multi-chromophore system may be ensured by any convenientconnection. Terms such as “connected,” “attached,” “linked” andconjugated are used interchangeably herein and encompass direct as wellas indirect connection, attachment, linkage or conjugation unless thecontext clearly dictates otherwise. Any convenient connection,attachment, linkage or conjugation of the signaling chromophore inenergy-receiving proximity to the light harvesting multichromophore mayprovide for optical amplification via Förster energy transfer. Thesignaling chromophore may be connected directly or indirectly to thelight harvesting multichromophore. In some embodiments, the signalingchromophore and the light harvesting multichromophore may be connectedin a detection reagent prior to assaying a sample for the presence of atarget.

In some cases, the connection between the light harvestingmultichromophore and the signaling chromophore is indirect, e.g., isachieved by virtue of a binding event. In certain instances, theconnection between the light harvesting multichromophore and thesignaling chromophore is direct, e.g., via covalent bonds. In someinstances, the connection between the light harvesting multichromophoreand the signaling chromophore is direct and is achieved by virtue of adirect linkage or conjugation. In certain embodiments, the lightharvesting multichromophore and the signaling chromophore may beattached to a sensor molecule.

Aspects of the invention include labelling compositions that include asignaling chromophore and a light harvesting multichromophore. As usedherein, the terms “labelling composition” and “detection reagent” areused interchangeably, and refer to a composition that finds use inlabelling and detecting a target analyte. When the average distancebetween the light harvesting multichromophore and the signalingchromophore is too large for effective energy transfer, there is littleor no emission from the signaling chromophore. As such, the lightharvesting multichromophore may be configured upon excitation totransfer energy to, and amplify the emission from, an acceptor signalingchromophore in energy-receiving proximity therewith.

It is understood that any convenient components and methods may beutilized in connecting the light harvesting multichromophore and thesignaling chromophore to provide a labelling composition, as long asthose components and methods permit energy transfer from the lightharvesting multichromophore to the signaling chromophore in thelabelling composition, for example via the Förster energy transfermechanism. In some cases, the connection between the light harvestingmultichromophore and the signaling chromophore is achieved where asignaling chromophore is conjugated to a sensor molecule that is alsoassociated with a light harvesting multichromophore, where the sensormolecule has affinity for the target analyte. Under these circumstances,the connection between the signaling chromophore and the lightharvesting multichromophore system is achieved, leading to efficientenergy transfer and intense emission from the signaling chromophore. Assuch, it is understood that the signaling chromophore should be inenergy-receiving proximity with the light harvesting multichromophore toachieve optical amplification in the labelling composition. Suchenergy-receiving proximity may be achieved by virtue of any convenientdirect or indirect connection. It is understood that the target analytemay be labelled with the light harvesting multichromophore and anacceptor signaling chromophore in energy-receiving proximity therewith.

The subject compositions including a signaling chromophore and a lightharvesting multichromophore may further include one or more components.In some cases, the composition includes a signaling chromophore, a lightharvesting multichromophore and a sensor molecule that specificallybinds a target.

In some instances, the invention provides a predominantly aqueoussolution including a light harvesting multichromophore, a sensormolecule and a signaling chromophore. In certain embodiments, the sensormolecule is conjugated to the signaling chromophore. In certain cases,the sensor molecule is conjugated to the light harvestingmultichromophore. In certain cases, the signaling chromophore isconjugated to the light harvesting multichromophore. In certainembodiments, the light harvesting multichromophore is conjugated to thesignaling chromophore and the sensor molecule.

In some embodiments, the invention provides a predominantly aqueoussolution comprising a light harvesting multichromophore (e.g., a CP), a“sensor biomolecule” and a signaling chromophore. In certainembodiments, the light harvesting multichromophore is conjugated to thesignaling chromophore and the sensor molecule.

As discussed herein, the optical amplification provided by a watersoluble light harvesting multichromophore can be used to detect a targetanalyte. In some cases, the amplification can be enhanced by usinghigher molecular weight water soluble conjugated polymers or otherstructures as the light harvesting multichromophore. The invention canbe provided in a homogeneous format that utilizes the ease offluorescence detection methods. Amplification of the emission from theacceptor signaling chromophore may occur when incident light is at awavelength absorbed by the donor light harvesting multichromophoresystem as compared to when the acceptor signaling chromophore isdirectly excited by incident light, e.g., at the absorbance maximum ofthe acceptor. In certain embodiments, the amplification of the emissionfrom the acceptor signaling chromophore is 2-fold greater or more, suchas 3-fold greater or more, 4-fold greater or more, 6-fold greater ormore, 8-fold greater or more, 10-fold greater or more, 15-fold greateror more, 25-fold greater or more, 30-fold greater or more, or even moreas compared to when the acceptor signaling chromophore is directlyexcited by incident light.

In some embodiments of the compositions and methods, the ratio of thedonor light harvesting multichromophore to the acceptor signalingchromophore is 1:1. In certain instances, the donor light harvestingmultichromophore includes a conjugated polymer of aromatic repeat unitsand the ratio of the number of repeat units to the acceptor signalingchromophore is 100:1 or more.

Methods of Use

Aspects of the invention include contacting a sample with apredominantly aqueous composition including: (a) a light harvesting,luminescent multichromophore system such as, for example, a conjugatedpolymer, semiconductor quantum dot or dendritic structure that is watersoluble; and (b) a sensor molecule conjugated to a luminescent signalingchromophore C*. The emission of a wavelength of light characteristic ofthe signaling chromophore-C* upon excitation of the light harvestingmultichromophore may be used to detect the target. In some embodiments,the emission of the signaling chromophore is used to detect the sensormolecule-target complex.

The light harvesting multichromophores may be used in methods whichscreen the light harvesting multichromophores for any property ofinterest. For example, the light harvesting multichromophores may betested for energy transfer to a chromophore, for increased fluorescentefficiency, for decreased self-quenching, for absorbance wavelength,and/or for emission wavelength.

A sensor molecule that is specific for the target may be used inconjugation with the light harvesting multichromophores, as can asignaling chromophore to which energy may be transferred from the lightharvesting multichromophores. In certain instances, a sensor molecule ofknown structure is used to label the target in the sample. The sensormolecule may provide a signal specific to its complementary target inany of various ways, e.g., through incorporation of a specific signalingchromophore which can receive energy from the light harvestingmultichromophore (e.g. CP). The signaling chromophore may beincorporated into the sensor molecule, or in some cases, may berecruited to a complex formed from the sensor molecule and the target.Formation of such a complex results in an increase of energy transferfrom a light harvesting multichromophore upon excitation to thesignaling chromophore, which may be detected directly or indirectly toprovide information regarding the target.

The compositions described herein are useful for any assay in which asample can be interrogated regarding a target biomolecule. In someembodiments, the assays involve determining the presence of the targetin the sample or its relative amount, or the assays may be quantitativeor semi-quantitative. As such, provided are methods of determiningwhether a target in present in a sample. In some embodiments, the methodincludes: contacting the sample with: a labelling composition (e.g., asdescribed herein) to produce a labelling composition contacted sample;and assaying the labelling composition contacted sample for the presenceof fluorescently labeled target analyte to evaluate whether the targetanalyte is present in the sample. Also provided are methods of detectinga target in a sample. In some instances, the method includes: contactingthe sample with a labelling composition (e.g., as described herein) toproduce a labelled target; and detecting the labelled target. Themethods of the invention can all be performed in multiplex formats. Aplurality of different sensor biomolecules can be used to detectcorresponding different target biomolecules in a sample through the useof different signaling chromophores conjugated to the respective sensorbiomolecules. In some cases, the light harvesting multichromophore(e.g., as described herein) is connected to the sensor biomolecule toprovide for amplification of the signaling chromophore. Multiplexmethods are provided employing 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200,400 or more different sensor biomolecules which can be usedsimultaneously to assay for corresponding different target biomolecules.The subject methods and compositions can be performed or utilized on asubstrate, as well as in solution.

Any target molecule and any sensor molecule that can bind to each othercan in principle be used in conjunction with the subject methods, wherethe light harvesting multichromophore may be associated with the sensormolecule. In some cases, this attachment may be accomplished through anyconvenient means to configure the light harvesting multichromophore insignaling juxtaposition with the signaling chromophore. In certaininstances, the light harvesting multichromophore is attached to thesensor molecule in signaling juxtaposition to the signaling chromophore,where the sensor molecule specifically binds the target.

Targets

The target molecule may be a biomolecule, for example a peptide orprotein, a polynucleotide such as DNA or RNA, and an antibody.Similarly, sensor molecules of interest include biomolecules. Exemplarysensor biomolecules of interest include, but are not limited to, apolynucleotide, a peptide nucleic acid (PNA), an antibody, a peptide ora protein.

Samples

Where the target is present in a biological sample, the portion of thesample comprising or suspected of comprising the target can be anysource of biological material that can be obtained from a livingorganism directly or indirectly, including cells, tissue or fluid, andthe deposits left by that organism, including viruses, mycoplasma, andfossils. The sample may comprise a target prepared through syntheticmeans, in whole or in part. Typically, the sample is obtained as ordispersed in a predominantly aqueous medium. Nonlimiting examples of thesample include blood, urine, semen, milk, sputum, mucus, a buccal swab,a vaginal swab, a rectal swab, an aspirate, a needle biopsy, a sectionof tissue obtained for example by surgery or autopsy, plasma, serum,spinal fluid, lymph fluid, the external secretions of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva,tumors, organs, samples of in vitro cell culture constituents (includingbut not limited to conditioned medium resulting from the growth of cellsin cell culture medium, putatively virally infected cells, recombinantcells, and cell components), and a recombinant library comprisingpolynucleotide sequences.

The sample can be a positive control sample which is known to containthe target or a surrogate therefor. A negative control sample can alsobe used which, although not expected to contain the target, is suspectedof containing it (via contamination of one or more of the reagents) oranother component capable of producing a false positive, and is testedin order to confirm the lack of contamination by the target of thereagents used in a given assay, as well as to determine whether a givenset of assay conditions produces false positives (a positive signal evenin the absence of target in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target present or to render itaccessible to reagents, e.g., to detection reagents. Where the samplecontains cells, the cells can be lysed or permeabilized to release thetarget within the cells.

Substrates

The methods described herein can be performed on a substrate in any of avariety of formats. One or more of the assay components may beincorporated in, attached to, or otherwise associated with thesubstrate, directly or indirectly. The substrate can comprise a widerange of materials, such as biological, nonbiological, organic,inorganic, or a combination of any of these.

Excitation and Detection of the Chromophores

Any convenient instrument that provides a wavelength that can excite thelight harvesting multichromophore and is shorter than the emissionwavelength(s) to be detected can be used for excitation. The excitationsource in some cases does not significantly excite the signalingchromophore directly. The source may be: a broadband UV light sourcesuch as a deuterium lamp with an appropriate filter, the output of awhite light source such as a xenon lamp or a deuterium lamp afterpassing through a monochromator to extract out the desired wavelengths,a continuous wave (cw) gas laser, a solid state diode laser, or any ofthe pulsed lasers. The emitted light from the signaling chromophore canbe detected through any convenient device or technique. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of the signalingchromophore upon excitation of the multichromophore.

Kits

Kits comprising reagents useful for performing the methods of theinvention are also provided. In one embodiment, a kit comprises a sensormolecule (e.g., as described herein) that specifically binds to a targetmolecule of interest and a light harvesting multichromophore (e.g., asdescribed herein). The sensor molecule may be conjugated to a signalingchromophore. In certain instances, the sensor molecule is a protein. Incertain embodiments, the light harvesting multichromophore is aconjugated polymer (CP).

The signaling chromophore may be configured in energy receivingproximity to the light harvesting multichromophore. In the presence ofthe target in the sample, the sensor molecule binds to the target whichcan be detected. The fluorescence detected includes increased emissionof energy from the signaling chromophore.

The components of the kit can be retained by a housing. Instructions forusing the kit to perform a method of the invention can be provided withthe housing, and can be provided in any fixed medium. The instructionsmay be located inside the housing or outside the housing, and may beprinted on the interior or exterior of any surface forming the housingwhich renders the instructions legible. The kit may be in multiplexform, containing pluralities of one or more different sensor molecules(e.g., PNAs) which can bind (e.g., hybridize) to corresponding differenttarget molecules (e.g., target polynucleotides).

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete description of how to make and use thepresent invention, and are not intended to limit the scope of what isregarded as the invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessotherwise indicated, parts are parts by weight, temperature is degreecentigrade and pressure is at or near atmospheric, and all materials arecommercially available.

Example 1: PNA Sensor Molecules Example 1-1: Identification of aMultichromophore/Signaling Chromophore Pair for FRET

The ability to transfer energy from the light harvestingmultichromophore system to the signaling chromophore on a sensor PNA wasdemonstrated using the water soluble conjugated polymerpoly((9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene),polymer 1 with iodide counteranions, and the sensor peptide nucleic acidPNA-C* having the sequence 5′-CAGTCCAGTGATACG-3′ (SEQ ID NO: 10) andconjugated to fluorescein (C*) at the 5′ position. Excitation wasperformed at 380 and 480 nm for 1 and PNA-C*, respectively. The datashow that there is an optical window for the specific excitation ofpolymer 1. Moreover, there is excellent overlap between the emission ofpolymer 1 and the absorption of C* to allow FRET.

Example 1-2: Demonstration of Amplified Fluorescence Via FRET BetweenLight Harvesting Multichromophore and Signalling Chromophore in Complexwith Target

The PNA-C* probe ([PNA-C*]=2.5×10⁻⁸ M) was contacted with an equimolaramount of the complementary 15 base pair ssDNA, (5′-CGTATCACTGGACTG-3′)(SEQ ID NO: 1), and in an identical fashion with a non-complementary 15base ssDNA, (5′-ACTGACGATAGACTG-3′) (SEQ ID NO: 2), in separate vesselsin the absence of polymer 1. The annealing step was performed in theabsence of buffer, i.e. at low ionic strength, at 2° C. below the T_(m)of PNA-C* (72° C. at 10⁻⁸M, pH=5.5). A melting experiment was performedand the absorbance monitored by UV/Vis spectroscopy at 260 nm.Increasing the temperature led to an increase in absorbance upon meltingof the hybridized duplex in the sample containing the complementaryssDNA, as the two single strands absorb more highly than the hybridizedduplex. As expected, the sample containing the non-complementary ssDNAdid not show such an increase in absorbance, as no duplex was formed inthat sample.

FRET was measured in annealed samples containing the complementary andnon-complementary ssDNAs and polymer 1 ([1]=2.3×10⁻⁷M). The normalizedemission spectra of PNA-C* in the presence of complementary andnon-complementary DNA upon excitation of polymer 1 are shown in FIG. 1.A FRET ratio>11 times higher for the PNA/DNA hybrid was detected,relative to the non-complementary pair. The fluorescein emission wasmore than 8 times larger than that obtained from direct C* excitation inthe absence of 1. This increased C* emission in the energy transfercomplex indicates that optical amplification is provided by themultichromophore (polymer 1).

Example 2: Polynucleotide Sensor Molecules Example 2-1: Identificationof FRET Between Light Harvesting Multichromophore and SignallingChromophore

Energy transfer from the light harvesting multi-chromophore system tothe signaling chromophore was demonstrated using the water solubleconjugated polymerpoly(9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene),polymer 1 with iodide counteranions. The sensor polynucleotide sequencewas 5′-GTAAATGGTGTTAGGGTTGC-3′ (SEQ ID NO: 3), corresponding to theanthrax (Bacillus anthracis) spore encapsulation plasmid, pX02, withfluorescein at the 5′ position, forming an example of oligo-C*. The datashows an optical window for the excitation of polymer 1, between theabsorption of DNA and fluorescein. Direct excitation of polymer 1results in energy transfer (ET) to fluorescein. The absorbance overlapof fluorescein with the emission of polymer 1 was selected to ensureFRET. The extent of ET is evaluated by the ratio of integrated acceptorto donor emission.

Example 2-2: Demonstration of Amplified Fluorescence Via FRET BetweenLight Harvesting Multichromophore and Signalling Chromophore in aComplex with a Target

The sensor polynucleotide ([Oligo-C*]=2.1×10⁻⁸M) was annealed at 2° C.below its T_(m) (58.4° C.) in the presence of an equal molar amount of a40 base pair strand containing a complementary 20 base pair sequence,5′-CATCTGTAAATCCAAGAGTAGCAACCCTAACACCATTTAC-3′ (SEQ ID NO: 4), and in anidentical fashion with a non-complementary 40 base strand with thesequence 5′-AAAATATTGTGTATCAAAATGTAAATGGTGTTAGGGTTGC-3′ (SEQ ID NO: 5).Direct comparison of the resulting fluorescence reveals an ET ratiogreater than 6 fold higher for the hybridized DNA. Using a Xenon lampfluorometer, equipped with a standard PMT, the hybridized DNA providedover 3 fold greater ET ratios, at [sensor polynucleotide]=2.8×10⁻⁹M,than did the non-hybridized DNA.

Example 2-3: FRET Transfer to a Target Specific Dye

Experiments using 1 and ethidium bromide (“EB”) as a signalingchromophore demonstrate that direct energy transfer from 1 to EB couldbe shown in the presence of double-stranded DNA. The sensorpolynucleotide (5′-ATCTTGACTATGTGGGTGCT-3′) (SEQ ID NO: 6) lacking thesignaling chromophore ([Oligo]=1×10⁻⁸ M) was annealed at 2° C. below itsT_(m) (58.5° C.) in the presence of an equal molar amount of a 20 basepair strand containing a complementary 20 base pair sequence,(5′-AGCACCCACATAGTCAAGAT-3′) (SEQ ID NO: 7), and in an identical fashionwith a non-complementary 20 base pair strand with the sequence(5′-CGTATCACTGGACTGATTGG-3′) (SEQ ID NO: 8). The two DNA mixtures weremixed with Ethidum Bromide ([EB]=1.1×10⁻⁸M) at room temperature inpotassium phosphate monobasic-sodium hydroxide buffer solution (50 mM,pH=7.40) where the intercalation of the EB occurred within the duplexstructure of the hybridized DNA pair. Addition of polymer 1 in water([1]=1.6×10⁻⁷M) and subsequent excitation of 1 (380 nm) resulted inenergy transfer from 1 to the intercalated EB only in the case ofhybridized or double stranded DNA.

Example 3: Protein Sensor Molecules

The TAR RNA and dTAR RNA oligonucleotides described below were purchasedfrom Dharmacon Research Inc. (Lafayette, USA). The polypeptides modifiedby fluorescein on the N-terminus (Tat-C* and SH3-C*) were custom-made bySigma-Genosys (Texas, USA). All the fluorescence and FRET experimentswere carried out using a PTI Quantum Master fluorometer equipped with aXenon lamp excitation source in tris-EDTA buffer solution (10 mM,pH=7.4).

Example 3-1

Emission spectra and absorption spectra were obtained to show that thereis excellent overlap between the emission of polymer 1 and theabsorption of Tat-C* to ensure fluorescence resonance energy transfer.

Example 3-2

Emission spectra and absorption spectra were obtained to show that thereis excellent overlap between the emission of oligomer 1 and theabsorption of Tat-C* to ensure fluorescence resonance energy transfer,

Example 3-3

Emission spectra and absorption spectra were obtained to show that thereis excellent overlap between the emission of oligomer 2 and theabsorption of Tat-C* to ensure fluorescence resonance energy transfer.

Example 3-4

The Tat-C* probe ([Tat-C*]=1.0×10⁻⁸ M) was mixed with an equimolaramount of the TAR RNA at room temperature, and in an identical fashionwith a non-specific dTAR RNA. It is known that a bulge structure in TARRNA is a requirement for Tat peptide binding to TAR RNA. dTAR RNA isclosely related in structure to TAR RNA, lacking the three base bulgestructure necessary for Tat binding.

Addition of oligomer 1 in water ([oligomer 1]=8.0×10⁻⁸ M) and subsequentcomparison of the resulting fluorescence of Tat-C* obtained byexcitation at 365 nm reveals an intensity ratio>10 times higher for theTat-C*/TAR RNA, relative to the non-specific Tat-C*/dTAR RNA pair. Thefluorescein emission is more than 25 times larger than that obtainedfrom direct Tat-C* excitation at the absorption maximum of fluoresceinin the absence of oligomer 1. The increased Tat-C* emission in theenergy transfer complex indicates that optical amplification is providedby the conjugated oligomer 1.

Example 3-5

The Tat-C* probe ([Tat-C*]=1.0×10⁻⁸ M) was mixed with an equimolaramount of the TAR RNA at room temperature, and in an identical fashionwith a non-specific dTAR RNA. Addition of oligomer 2 in water ([oligomer2]=6.0×10⁻⁸ M) and subsequent comparison of the resulting fluorescenceof Tat-C* obtained by excitation at 375 nm reveals an intensity ratio 15times higher for the Tat-C*/TAR RNA, relative to the non-specificTat-C*/dTAR RNA pair. The fluorescein emission is more than 30 timeslarger than that obtained from direct Tat-C* excitation in the absenceof oligomer 2. The increased Tat-C* emission in the energy transfercomplex indicates that optical amplification is provided by theconjugated oligomer 2.

Example 3-6

The water soluble conjugated polymer 1 (average n=app. 15) was utilizedas the light harvesting chromophore. The Tat-C* probe([Tat-C*]=1.0×10⁻⁸M) was mixed with an equimolar amount of the TAR RNAat room temperature, and in an identical fashion with a non-specificdTAR RNA. Addition of polymer 1 in water ([polymer 1]=4.8×10⁻⁷M) intothe mixture of Tat-C* and TAR RNA results in fluorescence of Tat-C* withan intensity ratio>15 times higher than that of the non-specificTat-C*/dTAR RNA and 10 times larger than that obtained from directTat-C* excitation in the absence of polymer 1. Thus, significantlyhigher FRET ratios and correspondingly higher sensitivities can beachieved.

Example 3-7

Another peptide sequence labeled with fluorescein at the N-terminus(SH3-C*; AKPRPPRPLPVAC in single letter code) (SEC) ID NO: 9) whichcannot specifically bind to TAR RNA was also utilized as the signalmolecule. The mixture of [SH3-C* or Tat-C*]=1.0×10⁻⁸ M, [TARRNA]=1.0×10⁻⁸ M and [oligomer 1]=8.0) (10⁻⁸ M) shows C* emission onlywhen the Tat-C* was present.

Example 4

Conjugated polymers were synthesized through the Suzuki couplingreaction and a post-polymerization quarternization step. Syntheticexamples are given with respect to the specific polymers underFormula 1. The synthetic routes are shown in the Schemes below

An overview of the methods depicted in Schemes 1-4 is as follows.1,3-Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)phenylene (1) wasobtained in 46% yield by treating 1,3-diiodobenzene withbis(pinacolato)diborane in the presence of PdCl₂(dppf) and potassiumacetate in DMSO. 2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (2) wasobtained by the treatment of 2,7-dibromofluorene with 50% KOH, followedby addition of excess of 1,6-dibromohexane in 85% yield. Coupling of oneequivalent of the dibromide monomer with one net equivalent of diboronicacid or diboronic ester, under Suzuki coupling conditions usingPdCl₂(dppf) in refluxing THF/H₂O for 24 h, followed by purification gavethe desired polymers including bromo-substituted alkyl substituents in39% to 88% yield. The products were thoroughly washed with methanol andacetone, and then dried in vacuum for 24 h. Formation of thetrimethylamine derivatized polymers was achieved by stirring thepolymers in condensed trimethylamine in a THF/H₂O solvent mixture for 24h.

Example 4-1: 1,3-Bis(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan)phenylene(1)

A flask charged with 1,3-diiodobenzene (1.0 g, 3 mmol),bis(pinacolato)diborane (2.3 g, 9 mmol), potassium acetate (2.1 g, 21mmol), PdCl₂(dppf) (150 mg, 0.18 mmol), and 15 mL of anhydrous DMSO wasdegassed for 15 minutes. The mixture was stirred at 80° C. for 12 h,cooled to room temperature and then poured into 100 mL of ice water. Themixture was extracted with CHCl₃, and the combined organic layers weredried over anhydrous MgSO₄. After the solvent was evaporated, theresidue was purified by chromatography using silica gel(Hexane:CHCl3=1:1) and then recrystallized from ethanol to afford 1 (460mg, 46%) as a white solid. ¹H NMR (200 MHz, CDCl3): δ 8.28 (s, 1H),7.91-7.89 (d, 2H), 7.38 (t, 1H), 1.35 (s, 24H). ¹³C NMR (50 MHz, CDCl₃):δ 141.4, 137.8, 127.3, 83.9, 25.1.

Example 4-2: 2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (2)

To a mixture of tetrabutylammonium bromide (300 mg, 9.3 mmol), aqueouspotassium hydroxide (100 mL, 50%) and 1,6-dibromohexane (22.6 g, 92.6mmol) was added 2,7-dibromofluorene at 75° C. After 15 minutes, themixture was cooled down to room temperature, and extracted with CH₂Cl₂.The organic layer was washed with water, aqueous HCl, water and brine,dried over MgSO₄, and then concentrated. Unreacted 1,6-dibromohexane wasdistilled off. The residue was purified by silica gel columnchromatography (Hexane:CHCl₃=9:1) and recrystallized from ethanol togive 2 (4.8 g, 80%) as a white solid. ¹H NMR (200 MHz, CDCl₃): δ 7.2-7.4(m, 6H), 3.12 (t, 4H), 1.75 (t, 4H), 1.5 (m, 4H), 1.0 (m, 8H), 0.4 (m,4H). ¹³C NMR (50 MHz, CDCl₃): δ 152.3, 139.2, 130.5, 126.2, 121.7,121.4, 55.7, 40.2, 34.1, 32.8, 29.1, 27.9, 23.6.

Example 4-3: Poly(9,9-bis(6′-bromohexyl)fluorene-co-alt-1,3-phenylene)(M₁₀₀P₀)

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,3-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)phenylene (166 mg, 0.5mmol), Pd(PPh3)4 (8 mg) and potassium carbonate (830 mg, 6 mmol) wereplaced in a 25 mL round bottle flask. A mixture of water (3 mL) andtoluene (5 mL) was added to the flask. After degassing, the mixture wasrefluxed at 85° C. for 20 h, and then precipitated into methanol. Thepolymer was filtered and washed with methanol and acetone, and thendried in vacuum for 24 h to afford M₁₀₀P₀ (251 mg, 88%) as a lightyellow solid. 1H NMR (200 MHz, CDCl₃): δ 7.9-7.6 (m, 10H), 3.3-3.2 (t,4H), 2.1 (m, 4H), 1.7-1.6 (m, 4H), 1.3-1.2 (m, 8H), 0.8 (m, 4H). 13C NMR(50 MHz, CDCl₃): δ 152.1, 142.9, 140.9, 130.1, 129.5, 128.0, 126.8,122.5, 120.9, 55.9, 40.9, 34.5, 33.3, 29.7, 28.5, 24.3. GPC (THF,polystyrene standard), Mw: 40,250 g/mol; Mn: 14,980 g/mol; PDI: 2.8.UV-vis (CHCl₃): λmax=337 nm; PL (CHCl₃): λmax=363 nm.

Example 4-4: Poly(9,9-bis(6′-bromohexyl)fluorene-co-alt-1,4-phenvlene)(M₀P₁₀₀)

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,4-phenylenebisboronic acid (82.9 mg, 0.5 mmol), Pd(dppf)Cl₂ (7 mg) andpotassium carbonate (830 mg, 6 mmol) were placed in a 25 mL round bottleflask. A mixture of water (3 mL) and THF (6 mL) was added to the flaskand degassed. The mixture was refluxed at 85° C. for 24 h, and thenprecipitated into methanol. The polymer was filtered and washed withmethanol and acetone, and then dried in vacuum for 24 h to afford M0P100(220 mg, 78%) as an off-white solid. ¹H NMR (200 MHz, CDCl3): δ 7.8 (m,5H), 7.7-7.6 (m, 4H), 7.5 (m, 1H), 3.3 (t, 4H), 2.1 (m, 4H), 1.7 (m,4H), 1.3-1.2 (m, 8H), 0.8 (m, 4H). 13C NMR (50 MHz, CDCl3): δ 151.9,140.9, 140.7, 140.2, 128.1, 126.6, 121.8, 120.8, 55.7, 40.9, 34.5, 33.2,29.6, 28.3, 24.2. GPC (THF, polystyrene standard), Mw: 25,850 g/mol; Mn:12,840 g/mol; PDI: 2.0. UV-vis (CHCl3): λmax=372 nm; PL (CHCl3):λmax=408 nm.

Example 4-5: Random Copolymer M₂₅P₇₅

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,4-phenylenebisboronic acid (62.2 mg, 0.375 mmol),1,3-phenylenebisboronic acid (20.7 mg, 0.125 mmol), Pd(dppf)Cl2 (7 mg)and potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL roundbottle flask. A mixture of water (3 mL) and THF (6 mL) was added to theflask and degassed. The mixture was refluxed at 85° C. for 24 h, andthen precipitated into methanol. The polymer was filtered and washedwith methanol and acetone, and then dried in vacuum for 24 h to affordM₂₅P₇₅ (248 mg, 88%) as an off-white solid. ¹H NMR (200 MHz, CDCl₃): δ7.9-7.6 (m, 10H), 3.3-3.2 (t, 4H), 2.1 (m, 4H), 1.7-1.6 (m, 4H), 1.3-1.2(m, 8H), 0.8 (m, 4H). GPC (THF, polystyrene standard), Mw: 29,000 g/mol;Mn: 14,720 g/mol; PDI: 1.9. UV-vis (CHCl₃): λmax=365 nm; PL (CHCl₃):λmax=407 nm.

Example 4-6: Random Copolymer M₅₀P₅₀

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,4-phenylenebisboronic acid (41.5 mg, 0.25 mmol),1,3-phenylenebisboronic acid (41.5 mg, 0.25 mmol), Pd(dppf)Cl2 (7 mg)and potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL roundbottle flask. A mixture of water (3 mL) and THF (6 mL) was added to theflask and degassed. The mixture was refluxed at 85° C. for 24 h, andthen precipitated into methanol. The polymer was filtered and washedwith methanol and acetone, and then dried in vacuum for 24 h to affordM₅₀P₅₀ (220 mg, 78%) as an off-white solid. ¹H NMR (200 MHz, CDCl₃): δ7.9-7.6 (m, 10H), 3.3-3.2 (t, 4H), 2.1 (m, 4H), 1.7-1.6 (m, 4H), 1.3-1.2(m, 8H), 0.8 (m, 4H). GPC (THF, polystyrene standard), Mw: 17,340 g/mol;Mn: 10,080 g/mol; PDI: 1.7. UV-vis (CHCl₃): λmax=351 nm; PL (CHCl₃):λmax=405 nm.

Example 4-7: Random Copolymer M₇₅P₂₅

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,4-phenylenebisboronic acid (20.7 mg, 0.125 mmol),1,3-phenylenebisboronic acid (62.2 mg, 0.375 mmol), Pd(dppf)Cl₂ (7 mg)and potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL roundbottle flask. A mixture of water (3 mL) and THF (6 mL) was added to theflask. After degassing, the mixture was refluxed at 85° C. for 24 h, andthen precipitated into methanol. The polymer was filtered and washedwith methanol and acetone, and then dried in vacuum for 24 h to affordM₇₅P₂₅ (130 mg, 46%) as an off-white solid. ¹H NMR (200 MHz, CDCl₃): δ7.9-7.6 (m, 10H), 3.3-3.2 (t, 4H), 2.1 (m, 4H), 1.7-1.6 (m, 4H), 1.3-1.2(m, 8H), 0.8 (m, 4H). GPC (THF, polystyrene standard), Mw: 13,000 g/mol;Mn: 8,700 g/mol; PDI: 1.4. UV-vis (CHCl3): λmax=342 nm; PL (CHCl₃):λmax=400 nm.

Example 4-8: Random Copolymer M₉₀P₁₀

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (325 mg, 0.5 mmol),1,4-phenylenebisboronic acid (8 mg, 0.05 mmol), 1,3-phenylenebisboronicacid (75 mg, 0.45 mmol), Pd(dppf)Cl₂ (7 mg) and potassium carbonate (830mg, 6 mmol) were placed in a 25 mL round bottle flask. A mixture ofwater (3 mL) and THF (6 mL) was added to the flask and degassed. Themixture was refluxed at 85° C. for 24 h, and then precipitated intomethanol. The polymer was filtered and washed with methanol and acetoneand then dried in vacuum for 24 h to afford M₉₀P₁₀ (110 mg, 39%) as anoff-white solid. ¹H NMR (200 MHz, CDCl₃): δ 7.9-7.6 (m, 10H), 3.3-3.2(t, 4H), 2.1 (m, 4H), 1.7-1.6 (m, 4H), 1.3-1.2 (m, 8H), 0.8 (m, 4H). GPC(THF, polystyrene standard), Mw: 8,400 g/mol; Mn: 5,800 g/mol; PDI: 1.4.UV-vis (CHCl3): λmax=338 nm; PL (CHCl₃): λmax=400 nm.

Example 4-9: Derivatization of Conjugated Polymers

The following procedure may be adapted for use in the derivatization ofa conjugated polymer that includes bromo substituted alkyl groups.

Condensed trimethylamine (2 mL) was added dropwise to a solution of theneutral polymer M₁₀₀P₀ (60 mg) in THF (10 mL) at −78° C. The mixture wasallowed to warm up to room temperature. The precipitate was re-dissolvedby the addition of water (10 mL). After the mixture was cooled down to−78° C., extra trimethylamine (2 mL) was added and the mixture wasstirred for 24 h at room temperature. After removing most of thesolvent, acetone was added to precipitate M₁₀₀P₀+(63 mg, 78%) as a lightyellow powder. ¹H NMR (500 MHz, CD₃OD): δ 8.1-7.7 (m, 10H), 3.3-3.2 (t,4H), 3.1 (s, 18H), 2.3 (br, 4H), 1.6 (br, 4H), 1.3 (br, 8H), 0.8 (br,4H). ¹³C NMR (125 MHz, CD₃OD): δ 151.9, 142.4, 140.9, 140.6, 129.77,126.5, 126.1, 125.6, 121.6, 120.5, 66.7, 55.7, 52.5, 40.1, 29.2, 25.8,23.8, 22.6. UV-vis (H₂O): λmax=334 nm; PL (H₂O): λmax=369 nm. ε=3.69×10⁴M⁻¹ cm⁻¹ per monomer unit.

Example 4-10

UV-vis and fluorescence spectra for a range of compositions aresummarized in Table 3. There is a progressive blue shift in absorptionwith increasing meta content, consistent with the more effectiveelectronic delocalization across para linkages. The ε values are lowestfor polymers with intermediate compositions because the randomdistribution of conjugated segments results in broader absorption bands.

TABLE 3 Optical properties of polymers of interest. M_(n)P_(m) ⁺λ_(max), abS λ_(max), em ε^(a) Φ_(buffer) ^(b) M₁₀₀P₀+ 335 369 37 0.51M₉₀P₁₀+ 337 403 32 0.57 M₇₅P₂₅+ 347 410 30 0.50 M₅₀P₅₀+ 361 421 32 0.44M₂₅P₇₅+ 376 417 42 0.42 M₀P₁₀₀+ 384 417 46 0.42 ^(a)unit: 10³ Lcm⁻¹mol⁻¹^(b)50 mmol phosphate buffer, quinine bisulfite as the standard

Fluorescence spectra in water as a function of polymer composition.Increasing the para content past the 50:50 ratio does not perturb theemission maxima. Fast energy transfer, either by intra- or interchainmechanisms, localizes excitations on the longest conjugation segmentswithin the lifetime of the excited state. Table 3 shows that there islittle variation in the fluorescence quantum yields (Φ in Table 3).

Equation 1 describes how the FRET rate changes as a function of thedonor-acceptor distance (r), the orientation factor (κ), and the overlapintegral (J).

$\begin{matrix}{{k_{t{(r)}} \propto {\frac{1}{r^{6}} \cdot k^{2} \cdot {J(\lambda)}}}{J(\lambda)} = {\underset{0}{\overset{\infty}{o}}{F_{D}(\lambda)}{ɛ_{A}(\lambda)}\lambda^{4}{d\lambda}}} & (1)\end{matrix}$

Since M₅₀P₅₀+ and M₀P₁₀₀+ have similar emission frequencies, the valueof J using a common acceptor dye should be nearly identical between thetwo polymers. The fluorescence lifetimes of the two polymers are similar(400±50 ps). Therefore, differences in FRET efficiencies to a commonacceptor chromophore can extract information relevant to the averagepolymer/acceptor chromophore distance and the orientation of transitionmoments.

Example 4-11

FIG. 2 shoes a comparison of the intensity of signaling chromophore(e.g., EB (EB=Ethidium bromide)) emission from various compositionswhere a conjugated polymer and a signaling chromophore are connected viaa biomolecule, e.g., polymer/ds-DNA/EB in 50 mmol phosphate buffer(pH=7.4) with [ds-DNA]=1.0 E⁻⁸ M, [Polymer RU]=2.0 E⁻⁷ M, [EB]=1.1 E⁻⁶M. Emission intensity was normalized relative to the E value at theexcitation wavelength.

Although the invention has been described in some detail with referenceto the preferred embodiments, those of skill in the art will realize, inlight of the teachings herein, that certain changes and modificationscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention is limited only by the claims.

What is claimed is:
 1. A labelling composition, comprising: a donorlight harvesting multichromophore that is a conjugated polymer and uponexcitation is capable of transferring energy to an acceptor signalingchromophore; and the acceptor signaling chromophore that is afluorescent dye in energy-receiving proximity to the donor lightharvesting multichromophore.
 2. The labelling composition according toclaim 1, wherein amplification of the emission from the acceptorsignaling chromophore occurs when incident light is at a wavelengthabsorbed by the donor light harvesting multichromophore system than whenthe acceptor signaling chromophore is directly excited by incidentlight.
 3. The labelling composition according to claim 2, wherein theamplification of the emission from the acceptor signaling chromophore is2-fold greater or more.
 4. The labelling composition according to claim1, wherein the ratio of the donor light harvesting multichromophore tothe acceptor signaling chromophore is 1:1.
 5. The labelling compositionaccording to claim 1, wherein the donor light harvestingmultichromophore comprises a conjugated polymer of aromatic repeat unitsand the ratio of the number of repeat units to the acceptor signalingchromophore is 100:1 or more.
 6. The labelling composition according toclaim 1, wherein the donor light harvesting multichromophore is directlyconjugated to the acceptor signaling chromophore.
 7. The labellingcomposition according to claim 1, wherein the donor light harvestingmultichromophore is indirectly conjugated to the acceptor signalingchromophore.
 8. The labelling composition according to claim 1, furthercomprising a sensor biomolecule.
 9. The labelling composition accordingto claim 8, wherein the sensor biomolecule is linked to a substrate. 10.The labelling composition according to claim 1, wherein the donor lightharvesting multichromophore is linked to a substrate.
 11. The labellingcomposition according to claim 1, wherein the donor light harvestingmultichromophore comprises a semiconductor nanocrystal.
 12. Thelabelling composition according to claim 1, wherein the light harvestingmultichromophore is a conjugated polymer comprising a neutral conjugatedsegment having the structure:

wherein: R¹, R², R³, R⁴ and R⁵ are independently selected from the groupconsisting of alkyl, alkenyl, alkoxy, alkynyl, and aryl, alkylaryl,arylalkyl, and polyalkylene oxide, each optionally substituted, whichmay contain one or more heteroatoms, or may be not present; A, B, C, Dand I are independently selected from the group consisting of: hydrogen,SiR′R″R′″, N+R′R″R′″, a guanidinium group, histidine, a polyamine, apyridinium group, and a sulfonium group; E is selected from the groupconsisting of not present, —O—, —S—, —C(O)—, —C(O)O—, —C(R)(R′)—,—N(R′)—, and —Si(R′)(R″), wherein R′ and R″ are independently selectedfrom the group consisting of hydrogen, C₁₋₁₂ alkyl and C₁₋₁₂ alkoxy andC₃₋₁₀ cycloalkyl; and n is 1 to 10,000; wherein at least one of —R¹-A,—R²—B, —R³—C, —R⁴-D and —R⁵-I comprises a water soluble group (WSG). 13.The labelling composition according to claim 1, wherein the conjugatedpolymer is neutral.
 14. The labelling composition according to claim 8,wherein the sensor biomolecule is a target binding protein.
 15. Thelabelling composition according to claim 14, wherein the sensorbiomolecule is an antibody.
 16. The labelling composition according toclaim 12, wherein R³ and R⁴ are alkyl, each optionally substituted,which may contain one or more heteroatoms.
 17. The labelling compositionaccording to claim 12, wherein E and R⁵ are not present.